Modeling and rescue strategies for retinal ciliopathies using stem cells: Many retinal diseases are characterized by defects in cilia including, LCA, JSRD, and Usher syndrome. CEP290 is important for cilia biogenesis and transport. Ciliary functions of patients can be differentially affected due to distinct mutations in CEP290. We modeled LCA and JSRD pathogenesis and explore different therapeutic interventions using fibroblasts of mouse model, i.e., rd16, and patients, as well as iPSC-derived 3-D retinal organoids. Using human iPSC lines from fibroblasts of LCA and JSRD patients with mutations in CEP290 and NPHP5 and their familial controls, we have generated retinal organoids and collected neural retina at different stages of differentiation for immunohistochemistry, transmission electron microscopy and transcriptome analysis. CEP290-LCA patient iPSC-derived photoreceptors displayed defects in ciliogenesis, including minimal development of outer-segment like structure and mis-localization of rhodopsin. Our studies thus recapitulate the pathologic changes in CEP290-LCA patients and should serve as a useful model to test treatment strategies. We have attempted rescue of CEP290 function by delivery of a variety of functional domains of CEP290 into CEP290-LCA iPSC-derived organoids. Our first attempt focused on the myosin tail of CEP290 into patient organoids with a nonsense mutation (c.5668G>T p.G1890X), which leads to an in-frame deletion in the myosin tail of endogenous CEP290. We are also designing experiments to deliver other CEP290 segments into patient organoids to investigate their complementary effects with endogenous truncated proteins on early ciliogenesis of photoreceptors. Drug discovery and small Molecule screening using in vivo mouse retina and retinal organoids: Utilizing a high-throughput screening system in collaboration with NCATS, two candidate small molecules have been identified. In the initial experiments, each compound was injected at postnatal day (P) 4 and the retina were harvested and processed at P14. No toxicity was observed in either compound. One compound showed borderline effect in improving photoreceptor development. We are now doing dose escalation studies. As an alternative approach, we are also testing 1 drug candidate to reduce Nrl expression in a Nrl reporter mouse line, so as to generate dysfunctional rods to maintain cone photoreceptor survival. Modeling LCA caused by CRX mutations: Mutations in CRX which encodes an essential transcription factor for photoreceptor development can cause LCA. We have derived human iPSCs from CRX-LCA patients and familial controls and differentiated them toward photoreceptors. Retinal organoids of both patient and control lines have been generated. Immunohistochemical and transcriptome analyses showed deformed inner segment structures and delayed expression of markers of photoreceptor development, implying compromised maturation of photoreceptors derived from CRX-LCA patient iPSCs. Initial studies were conducted to test the feasibility of AAV vectors for recovery from CRX-LCA related defects. Qualitative analysis indicated higher expression of GFP with cultures transduced with the AAV2/2 serotype. Subsequent experiments delivering a vector containing human CRX cDNA with its native promotor region utilized the AAV2/2 serotype. Organoids derived form CRX-LCA patient iPSCS were exposed to AAV vectors at DD120. 4 weeks post-treatment, transduced organoids demonstrated qualitatively greater CRX and Rhodopsin expression which was nearly absent in untreated controls. Upregulated expression of visual arrestin, S opsin, and M/L opsin in transfected organoids was observed indicating that induced expression of the correct copy of CRX can partially rescue the molecular defects observed in photoreceptors derived from CRX-LCA iPSC patients. Analyzing transcriptome of retinal organoids derived from familial control iPSCs and optimizing retinal organoid formation: The iPSC technology has been leveraged for human retinal disease modeling; however, protocols, efficiency, and success rate are highly variable. To elucidate the role of these differences, the transcriptomes of retinal organoids derived via 3 protocols, 3 individuals, and 4 pluripotent stem cell lines have been analyzed. Results indicate distinct differentiation rates of various organoids when compared to previously published fetal retina and adult data. To expedite organoid differentiation, we altered the supplementation during organoid formation. We hypothesized that the 9CRAL supplementation in lieu of ATRA could accelerate ciliogenesis and outer segment formation. Our analyses demonstrated faster maturation of photoreceptors with outer segment-like formation observed as early as DD90 versus DD120 in ATRA-supplemented organoids. Examining the development process of mouse in vivo retina development and in vitro mouse organoid differentiation, other supplements including docosahexaenoic acid (DHA) and fibroblast growth factor 1 (FGF1 or aFGF) were identified that could accelerate and extend culture length of mouse and human in vitro retinal organoids. Mouse retinal organoids demonstrated increased expression of rod-specific, cone-specific, and synapse-associated genes as well as enhanced mitochondrial biogenesis. Coculture and use of Bioreactors: Modeling of retinogenesis in vitro is hampered by limited development of outer segments essential to detection of light by photoreceptors. We have used human pluripotent stem cell lines to differentiate RPE-like cells. Our differentiated RPE cells correctly express proteins similar to mature RPE cells, have polarity typical of RPE cells, and display distinct RPE morphology. In collaboration with Pete Lelkles, PhD, our lab has cultured RPE cells derived from iPSCs on electrospun and electroblown nanofibrous scaffolds composed of poly(caprolactone) (PCL) and soy protein. Current protocols for human retinal organoid formation and culture require a long-term culture period (>120 days) limiting their applicability for drug discovery and cell therapies. Mouse organoid formation matures more rapidly, with similar maturation occurring within 3 weeks. However, mouse organoids undergo necrosis around DD30 with typical protocols. We have designed a rotary bioreactor cell culture system to improve nutrient exchange and promote spheroid formation. This work is in progress. Non humam primate models of retinogenesis: Non-human primates (NHP) have retina anatomy and disease pathophysiology similar to humans. Culturing photoreceptors derived from NHP could provide a line of in vitro models not currently available for pre-clinical cell therapy trials. We are developing a protocol to form 3D retinal organoids derived from Rhesus macaque iPSCs (RMiPSCs). Organoids have lamination indicative of photoreceptor maturation with upregulated expression of photoreceptor-specific markers.